1. Field of the Invention
The present invention relates generally to methods for production of transforming growth factor .beta. and, more particularly, to methods for the large scale fermentation and purification of transforming growth factor .beta. from mammalian cell culture.
Transforming growth factor .beta. (TGF-.beta.) is a multi-functional peptide shown to be active in regulating a wide variety of both normal and neoplastic cell types. It is a 25,000 MW homodimer consisting of two 12,500 subunits bound together by nine disulphide bridges and is synthesized as a 391 amino acid molecule comprised of a 29 amino acid leader peptide and a 362 amino acid latent precursor. Mature, active TGF-.beta. consists of the C terminal 112 amino acids of the latent peptide. The precise means of physiological activation is unknown, and the mature peptide is not glycosylated. There are, however, three potential N-linked glycosylation sites in the precursor portion of the protein, all of which appear to be used.
At least five different TGF-.beta.'s have been described, including TGF-.beta. 1,2,3, and 4. The sequence homology between the different forms of the mature TGF-.beta. peptide ranges from 64-82%. The sequence homology between the precursor sequences is somewhat lower averaging about 40%. All are functionally homologous although there is some difference in TGF-.beta. receptor binding properties.
TGF-.beta. has been shown to be an effective cell growth promoter, particularly with epithelial cells, and the use of TGF-.beta. as a wound healing agent has been demonstrated.
It would therefore be desirable to provide methods for producing TGF-.beta. in relatively large quantities. It would be particularly desirable to provide methods for both fermentation and purification of TGF-.beta. from mammalian cell culture. The fermentation procedures should be able to produce large quantities of TGF-.beta., preferably at least 1 mg/L-day, and the purification procedures should be able to purify such quantities to a very high degree, preferably 99% purity or above.
2. Description of the Background Art
Mature TGF-.beta. has been purified on a laboratory scale from the conditioned media of producer cell lines. A six step purification procedure including lyophilization, acid resuspension, gel filtration, reverse phase high pressure liquid chromatography (HPLC), SDS-polyacrylamide gel electrophoresis, and extraction is described in Massague (1984) J. Biol. Chem. 259:9756-9761. An eight step purification procedure including lyophilization, acid extraction, dialysis, lyophilization, gel filtration, cation exchange HPLC, and reverse phase HPLC is described in Van den Eignden-Van Raaij et al. (1989) Biochem. J. 257:375-382. Mature TGF-.beta. has also been purified from several tissues and whole cells, generally employing a four step process including extraction with acid and ethanol, gel filtration, cation exchange, and reverse phase HPLC. See, e.g., Assoian et al. (1983) J. Biol. Chem. 258:7155-7160; Frolik et al. (1983) Proc. Natl. Acad. Sci. USA 80:3676-3680; and Roberts et al. (1983) Biochem. 22:5692-5698. Copending application Ser. No. 97/184,519 describes a fermentation system similar to that employed in the present invention.